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Phytophthora cinnamomi Rands, a soil-borne oomycete plant pathogen, is a major threat to natural vegetation in many countries, especially Australia. The disease that it causes also has economic impacts on several agricultural and forestry systems worldwide. To investigate the virulence mechanisms of P. cinnamomi the roles of elicitins and elicitin-like proteins were examined using a susceptible model species, Lupinus angustifolius. Elicitins are 10 kDa proteins secreted by many Phytophthora species. Here, elicitins were isolated from P. cinnamomi liquid culture and separated according to their isoelectric point. A β-cinnamomin elicitin was then purified and used to develop an immunoaffinity purified antibody as a tool to examine virulence. The specificity of the β-cinnamomin antiserum was confirmed through Western-blot analysis. β-cinnamomin was found to be produced at different P. cinnamomi life stages and in inoculated plant roots by using immuno detection via confocal laser scanning microscopy. Pre-treatment of P. cinnamomi zoospores with β-cinnamomin antiserum and then inoculation of susceptible L. angustifolius roots revealed a partial loss of virulence of the pathogen. In addition, the expression of the β-cinnamomin gene at an early infection stage in susceptible L. angustifolius suggested an intrinsic role of the elicitin. Furthermore, the identification of covalently bound cell wall proteins in P. cinnamomi mycelia in this study has opened up the opportunity to investigate their role in virulence.

2082-09-15 Embargo por política editorial A second generation antivenomics protocol, based on affinity chromatography, was compared with a previously (first generation) immunodepletion protocol using as a proof of principle the pan-African EchiTAb-Plus-ICP® IgG antivenom and the venoms of Echis ocellatus, Bitis arietans, and African spitting cobras. The antivenom showed qualitatively similar immunoreactivity patterns using either antivenomic approach. Quantitative departures were noticed between both methods, which may be ascribed to differences in calculating the relative amounts of the non-recognized venom proteins. The smoother baseline in chromatograms of the affinity column allowed better resolution and more accurate quantification of the antivenomic outcome than the original immunodepletion protocol. Our results indicate that both methods can be used interchangeably to investigate the in vitro immunoreactivity of antivenoms. However, advantages of the second generation antivenomics are the possibility of analyzing F(ab′)2 antivenoms and the reusability of the affinity columns. These features contribute to the generalization, economy and reproducibility of the method. Ministerio de Economía y Competitividad/[BFU2010-17373]/MINECO/España Generalitat Valenciana/[PROMETEO/2010/005]//España Universidad de Costa Rica/[741-A9-003]/UCR/Costa Rica Universidad de Costa Rica/[741-B0-519]/UCR/Costa Rica Consejo Superior de Investigaciones Científicas/2009CR0021/CRUSA-CSIC/España UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Proteomic analysis of plasma is challenging because of its large dynamic range, which prevents the detection of low abundance proteins. Immunodepletion of high abundance proteins, such as albumin and IgG, has emerged as a favored technology to overcome this problem; however its suitability in quantitative expression proteomics has not yet been adequately addressed. In this study, albumin and IgG immunodepletion was evaluated by ELISAs and the reproducibility of depletion was tested with 2-DGE. Depletion of plasma resulted in removal of 62+/-1.2% of the total protein, 93+/-1.4% of the albumin (0.43 microg/microL, residual), and 94+/-1.5% of the IgG (0.21 microg/microL, residual). These results were confirmed by immunoblotting. Computerized image analysis of 2-D gels using Progenesis SameSpots software revealed an enhancement in the number of visible spots (675-1325), with 10+/-6% inter-gel variability in spot density. LC-ESI-MS/MS identification of newly resolved protein spots further validated the procedure. An innovative application of the software employed led to identification of 11 proteins lost non-specifically during depletion. This study demonstrates the effectiveness of immunodepletion of albumin and IgG in quantitative 2-DGE-based differential analysis of plasma proteins.

Natural killer (NK) cells contribute to the initial nonspecific response to viral infection, and viruses exhibit a range of sensitivities to NK cells in vivo. We investigated the role of NK cells in infection of mice by mouse adenovirus type 1 (MAV-1) using antibody-mediated depletion and knockout mice. MAV-1 causes encephalomyelitis and replicates to highest levels in brains. NK cell-depleted mice infected with MAV-1 showed brain viral loads 8–20 days p.i. that were similar to wild-type control non-depleted mice. Mice genetically deficient for NK cells behaved similarly to wild-type control mice with respect to brain viral loads and survival. We conclude that NK cells are not required to control virus replication in the brains of MAV-1-infected mice.